IL-15, that activates natural (NK) cells, has potential applications in cancer immunotherapy. Based on rhesus macaques' experiments, we performed a first-in-human Phase I dose-escalation trial of Escherichia coli (E. coli) produced rhIL-15 with NCI as the sponsor, Dr. Kevin Conlon as the Principal Investigator (PI) and Dr. Thomas Waldmann as Study Chairman and IND holder. This study was completed with 5 patients having finished their course of therapy at 3.0 mcg/kg/day, 4 patients having entered at 1.0 mcg/kg/day and 5 patients at the 0.3 mcg/kg/day dose of 12 daily infusions. Following the bolus IV infusion of IL-15 the serum concentrations of IL-15 at 10 minutes following infusion ranged from 20,000 to 90,000 picograms/mL-levels sufficient to signal through the IL-2/IL-15R beta and common gamma receptors shared with IL-2. The pharmacokinetics of various parameters for the 3.0, 1.0 and 0.3 mcg/kg/day doses resulted in a Cmax of 47,800 +/- 18,300 picograms/mL, 15,900 +/- 19,000 picograms/mL and 12,060 +/- 350 pg/mL respectively. The half-lives (T 1/2) were very similar for the three dose levels 2.4 +/- 0.5 hours, 2.7 +/- 0.13 hours and 2.7 +/- 0.67 hours. With a bolus infusion of 3.0 mcg/kg/day there was a pattern of fever beginning 2 to 2.5 hours following the start of the rhIL-15 infusions, peaking reliably at the 3-hour timepoint. Rigors occurred at the 4-hour timepoint. Rigors and fever were also observed at the 1.0 mcg/kg/day dose but were less severe. Up to 50-fold increases of serum levels of inflammatory cytokines in particular IL-6, IL-8 and IFN-gamma were observed. There was minimal fever observed at the 0.3 mcg/day dose. These toxicities were associated with maximum elevations at 4 to 8 hours post-bolus infusion of IL-15 in the serum concentrations of inflammatory cytokines (e.g. IL-16, IL-1, IL-8, IL-10 and IFN-gamma). Two patients manifested dose-limiting toxicity (DLT) at both the 3.0 mcg/kg/day (hypotension) and at the 1.0 mcg/kg/day (abnormalities of liver function). However the 0.3 mcg/kg/day dose has proven to be without DLT in the 5 patients examined. Flow cytometry of peripheral blood lymphocytes revealed a dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes upon IL-15 administration, followed by influx and hyperproliferation yielding10-fold expansions of NK and gamma delta T cells that ultimately returned to baseline. The pharmacokinetics of IL-15 following bolus infusion, discussed above, are clearly not optimal with exceedingly high levels initially that may have caused the toxicity. Therefore 2 additional clinical trials were initiated and are reaching completion using Escherichia coli rhIL-15 in the treatment of patients with metastatic malignancy. A trial was initiated using continuous intravenous infusion of rhIL-15 in the Clinical Center of the NCI. The maximum tolerated dose was 2 mcg/kg/day administered for a 10-day period. Furthermore, a subcutaneous dosing strategy was performed in concert with the Cancer Immunotherapy Network (CITN). In this dose-escalation trial 3 mcg/kg/day Monday-Friday for 2 weeks has been sustained without dose-limiting toxicity. In both the CITN subcutaneous trial and the continuous intravenous infusion in the one to three days following the termination of the IL-15 administration there was a dramatic increase that in the case of the CIV was 30-fold in the number of circulating NK cells and an over 200-fold increase in the number of CD56bright CD16dim NK-cell subpopulation. The approaches involving IL-15 studied to date were based on the hypothesis that the host is making an immune response albeit inadequate to the tumor and that this could be augmented by the administration of an IL-15 containing agent. However these cytokines could also be used in drug combination where an additional co-administered drug provides the specificity directed toward the tumor. In particular, IL-15 could be used with tumor-directed monoclonal antibodies to augment their antibody-dependent cell-mediated cytotoxicity (ADCC). We demonstrated that IL-15 administered with the anti-CD20 monoclonal antibody, rituximab yielded a dramatic increase in the ADCC mediated anticancer efficacy when used in an immunologically intact syngeneic tumor model involving the mouse EL4 leukemia transfected with human CD20. In parallel augmented ADCC was also noted in the xenograft MET model of adult T-cell leukemia when given in association with the anti-CD52 monoclonal antibody alemtuzumab. These studies support a clinical trial involving alemtuzumab in the treatment of patients with acute adult T-cell leukemia. Although IL-15 may show efficacy in the treatment of metastatic malignancy in the human trials it is not optimal when used as a single agent for cancer therapy. A particular challenge is that there is only a low level expression of IL-15R alpha on resting DCs. Indeed the true IL-15 cytokine may not be an IL-15 monomer but rather may be considered as an IL-15R alpha/IL-15 heterodimeric cytokine. To address this issue of deficiency of IL-15R alpha the Waldmann group has entered into collaboration with George Pavlakis of the Vaccine Branch, NCI and the company, Admune Therapeutics, LLC to initiate a trial of hetIL-15 (IL-15/IL15R alpha) in the therapy of patients with metastatic malignancy. As an alternative strategy, agents are available that induce IL-15R alpha expression on DCs that could be given in conjunction with IL-15 to circumvent the problem discussed above with IL-15 when used in monotherapy. The combination of IL-15 with the agonistic anti-CD40 antibody FGK4.5 showed additivity/synergy in 3 murine tumor models. It was demonstrated in each of these models that the combination of IL-15 with anti-CD40 produced markedly additive effects when compared with either agent administered alone. The combination appeared to circumvent the problem of helpless CD8 T cells, wherein CD8 T cells produced are not tumor- antigen specific. The administration of either IL-15 or anti-CD40 alone did not augment the number of tumor specific tetramer positive CD8 T cells in the TRAMP-C2 model system. However the administration of the combination of IL-15 plus the agonistic anti-CD40 antibody was associated with a meaningful increase in the number of TRAMP-C2 tumor specific SPAS-1/SNC9-H8 tetramer-positive CD8 T cells. We plan to translate this observation by using IL-15 in concert with an agonistic antibody to CD40 in the treatment of patients with metastatic malignancy. Collectively the two decades long scientific odyssey that involved the discovery and development of IL-15 by the Waldmann Group has accelerated progress in the field of Immunobiology that focuses on the role of the common gamma cytokines in the normal regulation of NK and CD8 T cell hemostasis and in disorders in disregulation in disease states. Furthermore, the completed initial trial evaluating IL-15 in patients with metastatic malignancy has provided the Phase I goal information required for the initiation of a broad range of studies using IL-15 in clinical trials by intramural and extramural NCI supported groups such as the Cancer Immunotherapy Network (CITN).